Fig 1: OTUD1 negatively regulates protein levels of MAVS, TRAF3, and TRAF6.(A) Immunoblot analysis of phosphorylated IRF3 (p-IRF3) in HeLa cells transfected with shRNAs plasmids (shCON or shOTUD1), and then infected with SeV for 12 hr. (B) HEK293T cells were transfected with vector or Flag-OTUD1, together with HA-RIG1, or HA-MAVS, or HA-TBK1. The IFNß luciferase activity was analyzed after SeV infection. The data were shown as fold change normalized to that in cells without HA-RIG1/MAVS/TBK1. (C) HEK293T cells were transfected with increasing amounts of Myc-OTUD1. The levels of indicated proteins were analyzed by immunoblotting. The densitometry of the proteins (MAVS, TRAF3 and TRAF6) is presented relative to those of ß–actin, and quantification of relative protein levels was from three independent experiments. (D) Immunoblot analysis of the indicated proteins in the Otud1+/+ or Otud1-/- primary MEFs. (E-G) MAVS, TRAF3 and TRAF6 were immunoprecipitated from whole cell lysates of Otud1+/+ or Otud1-/- MEFs. Ubiquitination levels of MAVS (E), TRAF3 (F) and TRAF6 (G) were detected by immunoblotting by a specific Ub antibody. (H-J) HEK293T cells were transfected with Myc-OTUD1, together with Flag-MAVS (H), or Flag-TRAF3 (I), or Flag-TRAF6 (J). Cells were either untreated or pretreated with MG132 (20 µM) for 4 hr before the harvest. The levels of Flag-MAVS (H), Flag-TRAF3 (I) and Flag-TRAF6 (J) were detected by immunoblotting as indicated. *P<0.05, **P<0.01 and ***P<0.001 (unpaired t-test). Data are representative of three independent experiments.
Fig 2: RNA virus infection promotes the interaction between OTUD1 and Smurf1, and binding of Smurf1 to MAVS/TRAF3/TRAF6.(A) 2fTGH cells were infected with SeV (MOI = 3) for 10 hr. Endogenous OTUD1 protein was immunoprecipitated, and the indicated proteins were tested by immunoblotting. The whole cell lysates (WCL) were used as a positive control. (B) HepG2 cells were infected with SeV (MOI = 3) for 8 hr, and then were stained by Smurf1 and OTUD1 antibodies. Cell nuclei were stained by DAPI. The fluorescent images were captured with the Nikon A1 confocal microscope. (C) Mouse BMDMs were infected with SeV (MOI = 3) for the indicated time. Endogenous Smurf1 protein was immunoprecipitated, and the indicated proteins were tested by immunoblotting. (D) HepG2 cells were infected as (B), and then were stained by a Smurf1 antibody. MitoTracker Red CMXRos was used for staining for mitochondria. (E) HepG2 cells were infected with SeV (MOI = 3) for 4 and 8 hr, and then were stained by an OTUD1 antibody or MitoTracker Red CMXRos. Data are representative of three independent experiments.
Fig 3: The OTUD1-Smurf1 axis regulates MAVS/TRAF3/TRAF6 levels and IFNß induction during RNA virus infection.(A) Immunoblot analysis of the indicated proteins in the whole cell lysates of HepG2 cells infected with SeV (MOI = 3) for the indicated times. (B) 2fTGH cells were infected with SeV (MOI = 3) for the indicated times. Relative mRNA amounts of IFNß were determined by q-PCR. The data were shown as fold change normalized to that in uninfected cells. (C-E) Relative mRNA amounts of MAVS (C), TRAF3 (D) and TRAF6 (E) in 2fTGH cells with SeV infection were determined by q-PCR. The data were shown as (B). (F) HeLa cells were transfected with shCON or shSmurf1, and then infected with SeV (MOI = 3). The levels of the indicated proteins were examined by immunoblotting. (G) BMDMs from Otud1+/+ or Otud1-/- mice were infected with VSV (MOI = 3). The levels of the indicated proteins were examined by immunoblotting. (H) Otud1+/+ or Otud1-/- MEF cells were infected with SeV for indicated times. Relative mRNA amounts of IFNß were determined by q-PCR. The data were shown as fold change normalized to that in uninfected Otud1+/+ MEFs. NS, not significant (P>0.05); *P<0.05, **P<0.01 and ***P<0.001 (unpaired t-test). Error bars represent the mean and s.d., and all data are representative of three independent experiments.
Fig 4: RNA virus infection promotes NF-?B-dependent expression of OTUD1, and which upregulates levels of Smurf1.(A) Q-PCR analysis of OTUD1 mRNA expression in HT1080 (left) infected with VSV (MOI = 3) or 2fTGH (middle and right) infected with SeV (MOI = 3) or HSV (MOI = 3). The data were shown as fold change normalized to that in uninfected cells. (B) Q-PCR analysis of OTUD1 mRNA expression in Vero (left) and U3A (right) cells infected with VSV (MOI = 3). The data were shown as (A). (C) Q-PCR analysis of OTUD1 mRNA expression in 2fTGH cells pretreated with PDTC (NF-?B inhibitor, 10 µM) for 1 hr before VSV (MOI = 3) infection. The data were shown as fold change normalized to that in uninfected control cells. (D) Q-PCR analysis of Smurf1 mRNA expression in HT1080 (left) and 2fTGH (right) cells infected with VSV (MOI = 3). The data were shown as (A). (E) Immunoblot analysis of Smurf1 and OTUD1 proteins in the whole cell lysates from MEFs cells infected with SeV (MOI = 3) for the indicated times. (F) Otud1+/+or Otud1-/- MEFs were infected with SeV (MOI = 3) for the indicated times. The level of Smurf1 protein was determined by Immunoblotting. NS, not significant (P>0.05); *P<0.05, **P<0.01 and ***P<0.001 (unpaired t-test). Error bars represent the mean and s.d., and all data are representative of three independent experiments.
Fig 5: Both Smurf1 and the deubiquitinase activity of OTUD1 are required for OTUD1-mediated downregulation of MAVS/TRAF3/TRAF6 and inhibition of interferon antiviral response.(A-C) HEK293T cells were transfected with Flag-MAVS (A), or Flag-TRAF3 (B), or Flag-TRAF6 (C), together with or without shSmurf1 and Myc-OTUD1 as indicated. The whole cell lysates were analyzed by immunoblotting. (D-F) HEK293T cells were transfected with Flag-MAVS (D), or Flag-TRAF3 (E), or Flag-TRAF6 (F), together with or without shSmurf1 and Myc-OTUD1 as indicated. Immunoprecipitation and immunoblot analysis were carried out as indicated. (G) HEK293T cells were transfected with or without shSmurf1 and Myc-OTUD1 as indicated. After 48 hr, cells were infected with SeV for 12 hr. Relative mRNA expression of IFNß was determined by q-PCR. The data were shown as fold change normalized to that in the shCON group without Myc-OTUD1. (H) Immunoblot analysis of Smurf1, MAVS, TRAF3 and TRAF6 proteins in whole cell lysates of HEK293T cells transfected with empty vector, wild-type Flag-OTUD1, or catalytically inactive Flag-OTUD1 (CH). (I) Otud1-/- MEFs were transfected with empty vector, Flag-OTUD1, or Flag-OTUD1 (CH), and then infected with SeV for 12 hr. Relative IFNß mRNA expression was determined by q-PCR. The data were shown as fold change normalized to that in the uninfected control group. (J) HEK293T cells were transfected with empty vector, or Flag-OTUD1 wild type, or Flag-OTUD1 (CH), and then infected with VSV for 12 hr. Relative VSV viral RNA expression was analyzed by q-PCR. The data were shown as (I). *P<0.05 and **P<0.01, NS, not significant (P>0.05) (unpaired t-test). Error bars represent the mean and s.d., and all data are representative of three independent experiments.
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